Method for killing or inhabiting the growth of sporulating microorganisms with haloperoxidase-containing compositions

ABSTRACT

Methods and compositions are provided for killing or inhibiting the growth of yeast or sporular microorganisms by contacting the microorganisms, in the presence of a peroxide and chloride or bromide, with a haloperoxidase and at least one antimicrobial activity enhancing agent. Suitable antimicrobial activity enhancing agents include certain α-amino acids, and are preferably compounds of the formula: ##STR1## wherein R 1  is hydrogen, an unsubstituted, or hydroxy or amino substituted, straight or branched chain alkyl group having from 1 to 6 carbon atoms, or an unsubstituted, or hydroxy or amino substituted arylalky group having from 7 to 12 carbon atoms, and R 2  is hydrogen or a straight or branched chain alkyl group having from 1 to 6 carbon atoms. The significantly enhanced haloperoxidase antiyeast and antispore activities make the methods and compositions of the invention highly useful in the therapeutic or prophylactic antiseptic treatment of human or animal subjects and in in vitro applications for disinfection or sterilization of vegetative microbes, yeasts, and bacterial and fungal spores. Suitable haloperoxidases for use in the methods and compositions of the invention include eosinophil peroxidase (EPO) and myeloperoxidase (MPO). Representative antimicrobial activity enhancing agents of the invention include α-amino acids selected from the group consisting of glycine and the l- or d-enantiomers of alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof.

This application is a divisional of prior application Ser. No.08/343,781, filed on Nov. 22, 1994, now U.S. Pat. No. 5,451,402, whichin turn is a divisional of application Ser. No. 08/100,780, filed Aug.2, 1993, now U.S. Pat. No. 5,389,369, which in turn is acontinuation-in-part of application Ser. No. 07/660,994, filed on Feb.21, 1991, now abandoned, the benefit of the filing dates of which arehereby claimed under 35 U.S.C. 120.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for thekilling of yeasts and sporular forms of microbes. More particularly, thepresent invention relates to methods and compositions using acombination of an antimicrobial activity enhancing agent andhaloperoxidase to enhance microbicidal properties of the system.

BACKGROUND OF THE INVENTION

As disclosed in PCT application Publication Number WO 92/14484,haloperoxidases, such as myeloperoxidase and eosinophil peroxidase, maybe used to selectively bind to and, in the presence of peroxide andhalide, inhibit the growth of target microorganisms without eliminatingdesirable microorganisms of significantly damaging other components ofthe medium, such as host cells and normal flora, in the targetmicroorganism's environment. Myeloperoxidase and eosinophil peroxidasehave previously been known to exhibit microorganism killing activity innatural systems when presented with an appropriate halide cofactor (X⁻)and hydrogen peroxide as substrate (Klebanoff, 1968, J. Bacteriol.95:2131-2138). However, the selective nature of haloperoxidase bindingand the utility of these systems for therapeutic, research andindustrial applications has only recently been recognized. Due to thenewly discovered selective binding properties of haloperoxidases, when atarget microorganism, such as a pathogenic microorganism, has a bindingcapacity for haloperoxidase greater than that of a desiredmicroorganism, such as members of the normal flora, the targetmicroorganism selectively binds the haloperoxidase with little or nobinding of the haloperoxidase by the desired microorganism. In thepresence of peroxide and halide, the target bound haloperoxidasecatalyzes halide oxidation and facilitates the disproportionation ofperoxide to singlet molecular oxygen (¹ O₂) at the surface of the targetmicroorganism, resulting in selective killing of the targetmicroorganism with a minimum of collateral damage to the desiredmicroorganism or physiological medium. Thus, as disclosed in PCTapplication Publication Number WO 92/14484, myeloperoxidase andeosinophil peroxidase can be employed as antiseptics in the therapeuticor prophylactic treatment of human or animal subjects to selectivelybind to and kill pathogenic microorganisms with a minimum of collateraldamage to host cells and normal flora of the host.

The system may also be employed in disinfecting or sterilizingformulations for inhibiting the growth of target microorganisms invitro, particularly in applications where biomedical devices, such asbandages, surgical instruments, suturing devices, catheters, dentalappliances, contact lenses and the like, are antiseptically treated toinhibit the growth of target microorganisms without damage to host cellsof a subject when the biomedical device is subsequently utilized invivo. While the haloperoxidase antiseptic system disclosed in PCTapplication Publication Number WO 92/14484 has been found to be highlyeffectively in the treatment of pathogenic microbes, yeast and somespore forming microorganisms remain relatively immune to haloperoxidaseantimicrobial activity.

The spore stage of the microbial life cycle is characterized bymetabolic dormancy and resistance to environmental factors that woulddestroy the microbe in its vegetative stage. The earliest phase of sporegermination is characterized by swelling and a shift from dormancy toactive metabolism. Vegetative growth, e.g., sprouting, and ultimatelyreproduction follows.

Germination of bacterial endospores and fungal spores is associated withincreased metabolism and decreased resistance to heat and chemicalreactants. For germination to occur, the spore must sense that theenvironment is adequate to support vegetation and reproduction. Theamino acid l-alanine is reported to stimulate bacterial sporegermination (Hills, 1950, J Gen Microbiol 4:38; Halvorson and Church,1957, Bacteriol Rev 21:112). L-alanine and l-proline have also beenreported to initiate fungal spore germination (Yanagita, 1957, ArchMikrobiol 26:329).

Simple α-amino acids, such as glycine and l-alanine, occupy a centralposition in metabolism. Tranamination or deamination of α-amino acidsyields the glycogenic or ketogenic carbohydrates and the nitrogen neededfor metabolism and growth. For example, transamination or deamination ofl-alanine yields pyruvate which is the end product of glycolyticmetabolism (Embden-Meyerhof-Parnas Pathway). Oxidation of pyruvate bypyruvate dehydrogenase complex yields acetyl-CoA, NADH, H⁺, and CO₂.Acetyl-CoA is the initiator substrate for the tricarboxylic acid cycle(Kreb's Cycle) which in turns feeds the mitochondrial electron transportchain. Acetyl-CoA is also the ultimate carbon source for fatty acidsynthesis as well as for sterol synthesis. Simple α-amino acids canprovide the nitrogen, CO₂, glycogenic and/or ketogenic equivalentsrequired for germination and the metabolic activity that follows.

Zgliczxnski et al. (1968, European J. Biochem 4:540-547) reported thatmyeloperoxidase catalyzes the chloride-dependent oxidation of aminoacids by hydrogen peroxide to yield ammonia, carbon dioxide and analdehyde corresponding to the decarboxylated, deaminated amino acid, andStrauss et al. (1970, J Reticuloendothel Soc 7:754-761) postulated thatthe aldehydes so produced might serve as microbicidal agents. However,Paul et al. (1970, Infect Immun 2:414-418) reported that adding theα-amino acids glycine and l-alanine to a myeloperoxidase-hydrogenperoxide-chloride test system actually inhibited killing of Escherichiacoli. Furthermore, Klebanoff (1975, Semin Hemat 12:117-142) reportedthat 100 mM acetaldehyde was required for bactericidal action. Contraryto these published data, it has now been surprisingly discovered thatthe microbicidal action of haloperoxidases against yeast and sporularforms of microbes may be significantly enhanced by treating themicroorganisms with haloperoxidase in combination with certain α-aminoacids which serve as an antimicrobial activity enhancing agent.

SUMMARY OF THE INVENTION

In accordance with the present invention, methods and compositions areprovided for killing or inhibiting the growth of yeast or sporularmicroorganisms comprising contacting the microorganisms, in the presenceof a peroxide and chloride or bromide, with a haloperoxidase and atleast one antimicrobial activity enhancing agent. Suitable antimicrobialactivity enhancing agents include certain α-amino acids, and arepreferably compounds of the formula: ##STR2## wherein R₁ is hydrogen, anunsubstituted, or hydroxy or amino substituted, straight or branchedchain alkyl group having from 1 to 6 carbon atoms, or an unsubstituted,or hydroxy or amino substituted arylalky group having from 7 to 12carbon atoms, and R₂ is hydrogen or a straight or branched chain alkylgroup having from 1 to 6 carbon atoms. In one embodiment, the methodsand compositions of the invention may be used to kill yeast and sporularmicrobes in vitro, to disinfect or sterilize medical products ormaterials. In other embodiments, the methods and compositions can beemployed in the antifungal and antiyeast treatment of human or animalsubjects without eliminating desirable microorganisms or significantlydamaging host cells. It has been discovered that the antiyeast andantifungal spore activities of haloperoxidases are significantlyenhanced in the presence of certain α-amino acids. In the furtherpresence of peroxide and halide, the target bound haloperoxidasecatalyzes halide oxidation and facilitates the disproportionation ofperoxide to singlet molecular oxygen at the surface of the spore formingmicroorganism, resulting in killing of the target microorganism.Although it is likely that haloperoxidase activity will catalyze thedeamination, decarboxylation of a portion of the added α-amino acids toyield aldehydes, it is unlikely that such aldehydes significantlycontribute to microbicidal action at such low concentrations. It islikely that these α-amino acids exert a mild concentration-dependentcompetitive inhibition of microbicidal action by consuming a portion ofthe haloperoxidase generated hypochlorous acid and singlet molecularoxygen. However, the stimulating effect of these α-amino acids on yeastbudding, germination of sporulated microbes, and possibly accelerationof metabolism of vegetative microbes appears to labilize the microbes sotreated to the actions of haloperoxidases and thus greatly enhancemicrobicidal action.

The significantly enhanced haloperoxidase antiyeast and antisporeactivities make the methods and compositions of the invention highlyuseful in the therapeutic or prophylactic antiseptic treatment of humanor animal subjects and in in vitro applications for disinfection andsterilization of vegetative microbes, yeasts, and bacterial and fungalspores.

Suitable haloperoxidases for use in the methods and compositions of theinvention include eosinophil peroxidase (EPO) and myeloperoxidase (MPO).Representative antimicrobial activity enhancing agents of the inventioninclude α-amino acids selected from the group consisting of glycine andthe l- or d-enantiomers of alanine, valine, leucine, isoleucine, serine,threonine, lysine, phenylalanine, tyrosine, and the alkyl estersthereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is broadly directed to methods and compositionsfor the killing or inhibition of yeast and sporulated microorganismsusing a haloperoxidase and an antimicrobial activity enhancing agentwhich labilizes the yeast and spore forms of the microorganism tohaloperoxidase microbicidal activity. In the practice of the invention,yeast and spore forms of microorganisms are killed or inhibited bycontacting the microorganisms with amounts of a haloperoxidase and anantimicrobial activity enhancing agent, i.e., certain α-amino acids,which are effective in the presence of a peroxide and bromide orchloride, to inhibit the growth of or kill the microorganisms.

In one particularly preferred embodiment, the methods and compositionsof the invention are used as antiseptic agents exhibiting enhancedhaloperoxidase antispore and antiyeast activity against a broad range ofpathogenic microorganisms including bacteria and fungi. For use incontact with host tissue, the antiseptic systems are based on the use ofdioxygenating haloperoxidase enzymes which exhibit selective affinityfor pathogenic microorganisms. As such, high potency microbicidal actioncan be directed to the target microorganisms without associated hosttissue destruction or disruption of normal flora; i.e., the antisepticaction is selective and confined to the target microorganism.

When properly formulated, haloperoxidase-enhancer preparations can beemployed to disinfect and even sterilize materials and devices. Highpotency haloperoxidase-enhancer formulations can serve as in vitrodisinfecting or sterilizing preparations. By limiting the time period ofhydrogen peroxide availability, haloperoxidase-enhancer formulations canbe made sufficiently potent to insure disinfection and evensterilization of a material or device before contact with host tissue.Any potential toxicity to normal flora and host tissue associated withthe use of these high potency formulations will cease when peroxide isdepleted, and as such, the formulation-treated material or device can bebrought in contact with host tissue without additional washing todetoxification.

Representative compositions of the invention comprise (1) eosinophilperoxidase (EPO) and/or myeloperoxidase (MPO), (2) hydrogen peroxide (H₂O₂) or equivalent peroxide, or an oxidase for the generation of H₂ O₂,(3) a substrate for the oxidase, and (4) an antimicrobial activityenhancing agent, such as glycine or l-alanine.

In one presently preferred embodiment, the invention provides methodsand compositions for inhibiting the growth of yeast and sporularmicroorganisms in vitro, particularly in applications where biomedicaldevices, such as bandages, surgical instruments, suturing devices,catheters, dental appliances, contact lenses and the like, requiredisinfection or sterilization and where the device is to be subsequentlycontacted with host tissue. The methods and compositions of theinvention may also be used to treat or prevent infections by yeast orspore forming microorganisms in vivo.

Haloperoxidases useful in the present invention are defined ashalide:hydrogen peroxide oxidoreductases (e.g., EC No. 1.11.1.7 and ECNo. 1.11.1.10 under the International Union of Biochemistry) for whichhalide, i.e., chloride or bromide, is the electron donor or reductantand peroxide is the electron receiver or oxidant. Any haloperoxidasewhich catalyzes the halide dependent generation of singlet molecularoxygen from hydrogen peroxide and which exhibits selective binding totarget microorganisms may be used in the present invention. Presentlyparticularly preferred haloperoxidases, as demonstrated herein, includeeosinophil peroxidase (EPO), myeloperoxidase (MPO) and combinationsthereof. Inclusion of an antimicrobial enhancing agent, as described indetail herein, greatly increases the microbicidal capacity of theoxidase-haloperoxidase system against yeast and sporular microorganismssince it labilizes these forms to the microbicidal action of thehaloperoxidase system.

Antimicrobial activity enhancing agents of the invention are agents thatenhance the antimicrobial activity of the haloperoxidase antimicrobialsystem against yeast and sporular microorganisms, used at concentrationsthat do not produce adverse effects on the haloperoxidase activity ofthe system or undesirable effects in the environment of use of themethods and compositions. Presently preferred activity enhancing agentsof the invention include α-amino acid compounds of the formula: ##STR3##wherein R₁ is hydrogen, a straight or branched chain alkyl group havingfrom 1 to 6 carbon atoms, or an unsubstituted or hydroxy or aminosubstituted straight or branched chain arylalky group having from 7 to12 carbon atoms, and R₂ is hydrogen or a straight or branched chainalkyl group having from 1 to 6 carbon atoms. As used herein, amino acidsmay be in their acid form, as shown above, or may be in theirzwitterionic form represented by the formula: ##STR4## wherein R₁ and R₂having the meanings set forth above, and may be in either l- ord-enantiomeric configurations. Representative alkyl R₁ groups include,for example, methyl, hydroxymethyl, isopropyl, 2-isobutyl, 1-isobutyl,hydroxy ethyl and amino butyl groups. Representative arylalkyl R₁ groupsinclude, for example, tolyl and hydroxytolyl groups. Presentlyparticularly preferred alkyl R₂ groups include methyl and ethyl groups.Representative antimicrobial activity enhancing agents of the inventioninclude α-amino acids selected from the group consisting of glycine andthe l- or d-enantiomers of alanine, valine, leucine, isoleucine, serine,threonine, lysine, phenylalanine, tyrosine and the alkyl esters thereof.The presently most preferred antimicrobial activity enhancing agents areglycine and l-alanine.

The nature and thickness of the spore wall affords protection againstthe lethal action of singlet molecular oxygen and hypochlorous acid.With respect to fungal spores, α-amino acid induced spore germinationyields vegetative forms that are more susceptible to oxidants. Inaddition, it has been found that the antimicrobial activity enhancingagents of the invention also increase oxidase-haloperoxidase killing ofyeast vegetative forms, including Candida albicans (see Table 1, below).This phenomenon may be related to the α-amino acid-dependentacceleration of yeast growth and budding, and the increasedsusceptibility of such metabolically active forms to haloperoxidasekilling. One alternative possibility is that α-amino acids, or metabolicproducts thereof, act as a substrate for a fungal oxidase capable ofgenerating H₂ O₂. Another alternative possibility is that the aldehydeproducts of haloperoxidase-mediated α-amino aciddeamination-decarboxylation might induce germination and budding, orotherwise effect some vital process.

Since the antiseptic activity of the haloperoxidase compositions of theinvention involves the reaction of peroxide and bromide or chloride toform hypohalite, and the reaction of peroxide and hypohalite to formsinglet molecular oxygen, the activity of the compositions of theinvention is dependent upon the presence, at the site of antimicrobialactivity, of a suitable peroxide and halide. In some situations,peroxide (e.g., hydrogen peroxide) may be present at the site ofantimicrobial activity due, for example, to the activity of naturallyoccurring flora, and sufficient amounts of chloride may be present inthe physiological milieu to act as a cofactor in the conversionreaction. In these situations, no additional peroxide or halide need beadministered or included in the compositions of the invention. In othersituations, it may be necessary to additionally provide hydrogenperoxide and/or halide at the site of microbial treatment. Accordingly,the compositions of the invention may additionally comprise, if desired,a peroxide or agent capable of producing peroxide in vivo or in vitroand a halide.

Peroxides useful in the methods and compositions of the inventioninclude hydrogen peroxide, alkyl hydroperoxides of the formula:

    R--OOH

wherein R is a hydrogen or a short chain alkyl group having from 1 to 3carbon atoms, and inorganic peroxides, such as boroperoxide orureaperoxide. The oxidant activity of the organic peroxides generallydecreases with increasing R chain length, as follows:

    R+H>>CH.sub.3 >CH.sub.3 CH.sub.2 >CH.sub.3 (CH.sub.2).sub.2

The presently preferred peroxide for use in the compositions of theinvention is hydrogen peroxide. Hydrogen peroxide may also be madeavailable at the site of the antimicrobial activity by including in thecomposition an agent capable of producing hydrogen peroxide in vivo orin vitro. Particularly useful agents for this purpose include, forexample, oxidases, such as cholesterol oxidase, glucose oxidase andgalactose oxidase.

When hydrogen peroxide is directly included in compositions of theinvention for in vivo applications, the amounts employed are preferablydesigned to provide maximum disinfecting activity. Damage to host cellsand tissue and to normal flora is avoided by avoiding direct contactduring the period of high H₂ O₂ exposure. Accordingly, when included inliquid formulations, the compositions of the invention may comprise fromabout 1 nmol to about 10 μmol of hydrogen peroxide per ml of liquidcomposition, more preferably from about 5 nmol to about 5 μmol ofhydrogen peroxide per ml of liquid composition, and most preferably fromabout 10 nmol to about 1 μmol of hydrogen peroxide per ml of liquidcomposition. Agents capable of producing hydrogen peroxide in vivo,e.g., peroxide producing oxidases, are particularly useful for dynamiccontrol of the amounts of hydrogen peroxide present at the site ofantimicrobial activity. Such agents maximize antimicrobial activity ofthe composition by providing and maintaining a steady, low levelconcentration of H₂ O₂. Accordingly, the amount of such agents to beemployed will be highly dependent on the nature of the agent and theeffect desired, but will preferably be capable of producing a steadystate level of from about 1 pmol to about 1 μmol of hydrogen peroxideper ml of liquid per minute, depending on the type and concentration ofhalide available at the site of antimicrobial activity. When theformulation is to be used as a disinfectant-sterilizing solution, theoxidase and its substrate can be adjusted to provide relatively highsteady-state concentrations of H₂ O₂ lasting for the requiredsterilization period. The disinfection-sterilizing action is terminatedwith exhaustion of the oxidase substrate or relative to the rate ofoxidase degradation.

For antifungal purposes, the use of cholesterol oxidase, e.g., fromNocardia erythropolis, as a H₂ O₂ producing oxidase is presentlyparticularly preferred. Unlike prokaryotic bacteria, fungi synthesizesterols. In fact, the antifungal activity of amphotericin B is at leastin part dependent on binding to fungal membrane steroids, e.g.,ergosterol. Ergosterol is the predominant sterol constituent of mostfungi, but other sterols are present (Weete, 1973, Phytochemistry12:1843). Cholesterol oxidase from Nocardia erythropolis selectivelyoxidizes Δ⁵⁻ 3β-ols and 5α-3β-ols to the resulting ketones (Smith andBrooks, 1974, J Chromatography 101:373); e.g.,

    cholesterol+O.sub.2 -cholesterol oxidase→cholestenone+H.sub.2 O.sub.2

    ergosterol+O.sub.2 -cholesterol oxidase→ergostenone+H.sub.2 O.sub.2

This Nocardia oxidase is relatively heat stable and retains catalyticactivity at 50° C. It is active over a pH range of 4 to 9 with a maximumactivity at pH 7. It has a Michaelis constant (Km) of 1.4×10⁻⁵ mol/liter(Richmond, 1973, Clin Chem. 19:1350).

Haloperoxidases are fungicidal when presented with H₂ O₂ or coupled to aH₂ O₂ -generating oxidase. However, with cholesterol oxidase as theoxidase, oxidase-haloperoxidase fungal killing is greater than expectedfrom the generation of H₂ O₂ alone. This cholesterol oxidase-dependentincrease in fungicidal action may in part be related to disruption offungal membrane integrity resulting from oxidase depletion of fungalsteroids. Fungi might also synthesize an endogenous H₂ O₂ -generatingsterol oxidase

Suitable halides for use in the methods and compositions of theinvention may be bromide or chloride. The use, selection, and amount ofhalide employed in a particular application will depend upon variousfactors, such as the haloperoxidase used in the antiseptic composition,the desired therapeutic effect, the availability of peroxide and otherfactors. When the haloperoxidase is myeloperoxidase, the halide may bebromide or chloride. Since chloride is present in most physiologicalmedia at levels sufficient to be nonlimiting as the halide cofactor, anexternal source of chloride is generally not required. When an externalsource of chloride is desired, the amount of chloride employed willpreferably fall in the range of about 10 μmol chloride to about 150 μmolchloride per ml of solution to approximate physiological conditions.

When the haloperoxidase is eosinophil peroxidase, chloride is relativelyineffective as a cofactor, and accordingly, the preferred halide isbromide. When included in liquid compositions, the compositions of theinvention may comprise from about 1 nmol bromide to about 20 μmolbromide per ml of liquid composition, more preferably from about 10 nmolbromide to about 10 μmol bromide per ml of liquid composition, and mostpreferably from about 100 nmol bromide to about 1 μmol bromide per ml ofliquid composition.

The ratio of halide to peroxide is an important consideration informulating an effective microbicidal environment. Accordingly, inaddition to ensuring effective levels of halide and peroxide at thesitus of microbial attack, as described above, it is preferable topractice the methods of the invention at halide peroxide ratios thatprovide optimal microbicidal activity. For example, when thehaloperoxidase is MPO and the halide is Cl⁻, the ratio of Cl⁻ toperoxide is preferably maintained in the range of about 1 to about40,000 in the environment of microbicidal activity, more preferably fromabout 50 to about 40,000 and most preferably from about 200 to about40,000. When the halide is Br⁻, the ratio of Br⁻ to peroxide ispreferably maintained in the range of about 0.1 to about 4,000 in theenvironment of microbicidal activity, more preferably from about 0.5 toabout 2,000 and most preferably from about 1 to about 1,000.

The methods and compositions of the invention can be used to inhibit thegrowth of a broad spectrum of sporular microorganisms, preferably with aminimum of damage to normal flora. As used herein, "sporularmicroorganisms" is intended to include spore forms of bacteria or fungi.Spore forming microorganisms are well known, and include, for example,bacteria such as Bacillus sps. and Clostridium sps., and fungi such asAspergillis sps., Fusarium sps., Trichophyton sps. and the like.

As used herein, the term "normal flora" means bacteria which normallyreside in or on body surfaces of a healthy host at symbiotic levels.Normal flora include, for example, the lactic acid family of bacteria inthe mouth, intestine, or vagina of human subjects, e.g. Streptococcus(viridans) in the mouth, and Lactobacillus sp. (e.g., Tissier's bacillusand Doderlein's bacillus) in the intestines of breast-fed infants,external genitalia, anterior urethra and vagina. Microorganisms whichconstitute normal flora of a host are well known (e.g., see Principlesand Practice of Infectious Diseases, supra, New York, pp. 34-36 and161). It has been found that the haloperoxidases of the inventionselectively bind to many pathogenic bacteria and fungi in preferenceover normal flora. In in vivo applications, the host is preferablytreated with an amount of haloperoxidase which is ineffective toeliminate normal flora from the host. In in vitro applications fordisinfection-sterilization, sufficiently high concentrations ofhaloperoxidase can be employed to ensure complete killing of allvegetative and yeast forms. Under such conditions, damage to host tissueand normal flora is avoided by consumption of H₂ O₂ or the H₂ O₂-generating system prior to contact with the host tissue.

The compositions of the invention generally comprise amounts of ahaloperoxidase and of an antimicrobial activity enhancing agent whichare effective, in the presence of a peroxide and a halide to kill orinhibit the growth of yeast or sporular microorganisms. The compositionsmay be conveniently provided in a liquid carrier. Any liquid carrier maybe generally used for this purpose, provided that the carrier does notsignificantly interfere with the selective binding capabilities of thehaloperoxide or with enzyme activity. Alternatively, the compositionsmay be provided in solid form with activation on solubilization inliquid.

The compositions of the invention may additionally comprise peroxide oran agent capable of producing peroxide, such as an oxidase, as describedin detail above. The oxidase-haloperoxidase system lends itself toconstruction as a binary formulation. One part of the binary comprises asolution containing the oxidase, the haloperoxidase and theantimicrobial activity enhancing substance, e.g., glycine or l-alanine.The second part of the binary comprises a substrate for the oxidase,e.g., cholesterol in the case of cholesterol oxidase or molecularoxygen, O₂. The substrate may be provided, for example, in the form of asolid wafer. For sterilization of an article, e.g., a contact lens, thecholesterol wafer is placed in a sterilization chamber along with theitem to be sterilized. The cholesterol oxidase-haloperoxidase plusglycine or l-alanine solution is added to initiate sterilization. Thiscomposition may additionally comprise alcohol in order to facilitatecholesterol solubilization and utilization by the oxidase. This systemwill produce sustained microbicidal action as long as sufficientcholesterol is present to drive the reaction.

For in vivo applications, the antiseptic compositions can beadministered in any effective pharmaceutically acceptable form to warmblooded animals, including human and animal subjects, e.g., in topical,lavage, oral or suppository dosage forms, as a topical, buccal, or nasalspray or in any other manner effective to deliver active haloperoxidaseto a site of microorganism infection. The route of administration willpreferably be designed to obtain direct contact of the antisepticcompositions with the infecting microorganisms.

For topical applications, the pharmaceutically acceptable carrier maytake the form of liquids, creams, foams, lotions, or gels, and mayadditionally comprise organic solvents, emulsifiers, gelling agents,moisturizers, stabilizers, surfactants, wetting agents, preservatives,time release agents, and minor amounts of humectants, sequesteringagents, dyes, perfumes, and other components commonly employed inpharmaceutical compositions for topical administration.

Solid dosage forms for oral or topical administration include capsules,tablets, pills, suppositories, powders, and granules. In solid dosageforms, the compositions may be admixed with at least one inert diluentsuch as sucrose, lactose, or starch, and may additionally compriselubricating agents, buffering agents, enteric coatings, and othercomponents well known to those skilled in the art.

In another embodiment of the invention, the compositions of theinvention may be specifically designed for in vitro applications, suchas disinfecting or sterilization of medical devices, contact lenses andthe like, particularly where the devices or lenses are intended to beused in contact with a patient or wearer. For applications of this type,the compositions may be conveniently provided in the form of a liquid orfoam, and may be provided with emulsifiers, surfactants, bufferingagents, wetting agents, preservatives, and other components commonlyfound in compositions of this type. Compositions of the invention may beimpregnated into absorptive materials, such as sutures, bandages, andgauze, or coated onto the surface of solid phase materials, such asstaples, zippers and catheters to deliver the compositions to a site forthe prevention of microbial infection. Other delivery systems of thistype will be readily apparent to those skilled in the art.

Actual amounts of haloperoxidase and antimicrobial activity enhancingagents in the compositions of the invention may be varied so as toobtain amounts of haloperoxidase and antimicrobial activity enhancingagents at the site of treatment effective to kill vegetative as well asyeast and sporular microorganisms. Accordingly, the selected amountswill depend on the nature and site for treatment, the desired response,the desired duration of microbicidal action and other factors.Generally, when the haloperoxidase is myeloperoxidase, liquidcompositions of the invention will comprise at least 0.01 picomoles(pmol) of myeloperoxidase per ml of liquid composition, more preferablyfrom about 0.1 pmol to about 500 pmol of myeloperoxidase per ml ofliquid composition, and most preferably from about 0.5 pmol to about 50pmol of myeloperoxidase per ml of liquid composition. Similar dosages ofeosinophil peroxidase may be employed. Optionally, it may be desirablein some applications to include both eosinophil peroxidase andmyeloperoxidase in the same composition. Liquid compositions of theinvention will generally comprise at least 0.005 μmol/ml ofantimicrobial activity enhancing agents, i.e., α-amino acids such asglycine and alanine, and more preferably from 0.05 μmol/ml to 50 μmol/mlof such antimicrobial activity enhancing agent.

Other components, such as an oxidase for peroxide generation, substratefor the oxidase and halide may be included, as desired, as described indetail above. In addition, the components may be formulated in a singleformulation, or may be separated into binary formulations for latermixing during use, as may be desired for a particular application. Forsingle formulations, one required system component which is available atthe application site, such as halide, oxidase, prosthetic group for theoxidase, reducing substrate for the oxidase, or molecular oxygen ispreferably left out of the formulation to preclude premature reactionand exhaustion of system components.

As an illustrative example, a composition suitable for use as a contactlens solution may comprise from 1 to 20 pmol/ml of eosinophil peroxidaseand/or myeloperoxidase, from 0.1 to 1 μmol/ml of glycine, from 0.01 to10 units of glucose oxidase, and from 50 to 500 mEq/L of chloride with0.1 to 1 mEq/L bromide. The above composition is combined with from 1 to10 μmol/ml of glucose under anaerobic conditions and the completepreparation is kept anaerobic until used as a liquid disinfectant orsterilizing solution. Exposure to air, i.e., molecular oxygen, activatesthe disinfecting-sterilizing action of the formulation.

The foregoing may be better understood in connection with the followingrepresentative examples, which are presented for purposes ofillustration and not by way of limitation.

EXAMPLES Example 1 The Effect of l-Alanine on Oxidase-HaloperoxidaseKilling of Bacterial, Yeast and Fungal Spores

The effect of l-alanine on oxidase-haloperoxidase killing of bacterial,yeast and fungal spores was determined as follows. Incubation media wasprepared from 50 mM acetate buffer containing 0.1 unit (i.e., 4 μg)cholesterol oxidase from Nocardia erythropolis (prepared in accordancewith the procedure of Richmond, W., "Preparation and Properties of aCholesterol Oxidase from Nocardia sp. and Its Application to theEnzymatic Assay of Total Cholesterol in Serum," Clin. Chem.19(12):1350-1356 (1973), 20 pmol (2.8 μg) porcine MPO (ExOxEmis, Inc.,San Antonio, Tex. U.S.A., Lot#1899201) or 20 pmol (1.5 μg) porcine EPO(ExOxEmis, Inc., San Antonio, Tex., U.S.A., Lot#1929201), 100 mEq/LCl³¹, and 1 mEq/L Br⁻. Incubation mixtures were prepared by inoculatingthe incubation media with 1×10⁷ cells of Staph. aureus, Cand. albicans,and Asperg. fumigatus. The pH of the incubation mixtures was adjusted to7 with 50 mM MOPS buffer. Cholesterol in 8.5% ethanol was added to theincubation mixtures to a final concentration of 7 mM (7 μmol/ml). Thefinal volume of the incubation mixtures was 1 ml. The mixtures wereincubated for four hours at 22° C. and the microbes were then plated (S.aureus was plated on trypticase soy agar; C. albicans and A. fumigatuswere plated on Sabouraud's dextrose agar). After about 24 hours (about72-96 hours for A. fumigatus), the colony forming units (CFU) werecounted as a measure of the viability of the organisms. The results areshown in Table 1.

                  TABLE 1    ______________________________________    Effect of l-Alanine on Cholesterol Oxidase-Haloperoxidase    Microbicidal Action Against Staphylococcus aureus,    Candida albicans, and Aspergillus fumigatus Spores:                Cholesterol    Organism    Oxidase   Haloperoxidase                                      CFU    ______________________________________    Staph. aureus                None      None        19,400,000    Staph. aureus                0.1 Unit  None        29,000,000    Staph. aureus                0.1 Unit †                          None        29,200,000    Staph. aureus                0.1 Unit  20 pmol MPO 0    Staph. aureus                0.1 Unit †                          20 pmol MPO 0    Staph. aureus                0.1 Unit  20 pmol EPO 0    Staph. aureus                0.1 Unit †                          20 pmol EPO 0    Cand. albicans                None      None        1,460,000    Cand. albicans                0.1 Unit  None        1,380,000    Cand. albicans                0.1 Unit †                          None        1,580,000    Cand. albicans                0.1 Unit  20 pmol MPO 800,000    Cand. albicans                0.1 Unit †                          20 pmol MPO 0    Cand. albicans                0.1 Unit  20 pmol EPO 680,000    Cand albicans                0.1 Unit †                          20 pmol EPO 0    Asperg. fumigatus                None      None        1,260,000    Asperg. fumigatus                0.1 Unit  None        1,020,000    Asperg. fumigatus                0.1 Unit †                          None        880,000    Asperg. fumigatus                0.1 Unit  20 pmol MPO 550,000    Asperg. fumigatus                0.1 Unit †                          20 pmol MPO 0    Asperg. fumigatus                0.1 Unit  20 pmol EPO 840,000    Asperg. fumigatus                0.1 Unit †                          20 pmol EPO 0    ______________________________________     †indicates that the 50 mM Acetate Buffer contained 1 mM lalanine.

As shown in Table 1, cholesterol oxidase plus either MPO or EPO providesa potent microbicidal system. This combination killed 10⁷ Staphylococcusaureus in the presence or absence of 1 mM l-alanine. However, inclusionof l-alanine in the cholesterol oxidase-haloperoxidase system wasnecessary for complete killing of both Candida albicans yeast forms andAspergillus fumigatus spores.

Example 2 Effect of Potential Amino Acid Antimicrobial ActivityEnhancing Agents on Haloperoxidase Microbicidal Action AgainstAspergillus fumigatus Spores

The effect of various potential amino acids as enhancing agents forhaloperoxidase microbicidal action was determined by following theprocedure of Example 1, except that each test contained the quantity ofglucose oxidase indicated in Tables 2-8, below (instead of cholesteroloxidase as in Example 1), in an incubation medium of 5.6 mM glucose in50 mM sodium acetate buffer containing 100 mEq/L of chloride and 0.1mEq/L of bromide at pH 6. The potential amino acid activators indicatedin Tables 2-8 below were added to the mixtures to a final concentrationof 0, 5, 0.5 or 0.05 μmol/ml, and the incubation mixtures wereinnoculated with about 1×10⁷ spores of Aspergillus fumigatus. Themixtures were incubated at ambient temperature for 90 minutes and thenplated as described in Example 1. The plates were grown overnight at 35°C., and then for an additional two days. The colony forming units werecounted as a measure of viability of the organisms.

The aliphatic amino acids glycine, l-alanine, l-valine, l-leucine andl-isoleucine were tested as described above. The results are shown inthe following Table 2:

                                      TABLE 2    __________________________________________________________________________    Amino Acid Type and Concentration:    Effect on Haloperoxidase Killing of Aspergillus fumigatus Spores            Glucose                 (Amino Acid)                         CFU (Aliphatic Amino Acids)    Haloperoxidase            Oxidase                 μmol/ml (mM)                         Glycine                              l-Alanine                                   l-Valine                                        l-Leucine                                             l-Isoleucine    __________________________________________________________________________    0       0    0       920,000                              800,000                                   260,000                                        920,000                                             260,000    0       0    5       560,000                              520,000                                   380,000                                        740,000                                             340,000    0       0    0.5     700,000                              660,000                                   460,000                                        960,000                                             400,000    0       0    0.05    480,000                              520,000                                   180,000                                        460,000                                             460,000    0       0.6 Units                 0       760,000                              1,140,000                                   420,000                                        740,000                                             420,000    0       0.6 Units                 5       440,000                              980,000                                   440,000                                        1,000,000                                             340,000    0       0.6 Units                 0.5     300,000                              780,000                                   300,000                                        920,000                                             400,000    0       0.6 Units                 0.05    580,000                              760,000                                   340,000                                        700,000                                             520,000    20 pmol MPO            0.6 Units                 0       500,000                              700,000                                   300,000                                        1,640,000                                             300,000    20 pmol MPO            0.6 Units                 5       0    0    34,000                                        72,000                                             22,000    20 pmol MPO            0.6 Units                 0.5     0    0    2,000                                        42,000                                             0    20 pmol MPO            0.6 Units                 0.05    260,000                              4,000                                   16,000                                        62,000                                             10,000    20 pmol EPO            0.6 Units                 0       780,000                              840,000                                   200,000                                        1,060,000                                             200,000    20 pmol EPO            0.6 Units                 5       0    0    0    52,000                                             0    20 pmol EPO            0.6 Units                 0.5     0    0    0    0    0    20 pmol EPO            0.6 Units                 0.05    182,000                              10,000                                   28,000                                        0    30,000    __________________________________________________________________________

As shown in Table 2, each of the aliphatic amino acids tested exhibiteda significant enhancing effect on the haloperoxidase antimicrobialactivity of both eosinophil peroxidase and myeloperoxidase against A.fumigatus spores, with glycine and l-alanine exhibiting the greatestenhancing effect.

The dicarboxylic amino acids and amides l-aspartic acid, l-asparagine,l-glutamic acid and l-glutamine, and the imino acids l-proline andl-hydroxyproline were tested as described above. The results are shownin the following Table 3:

                                      TABLE 3    __________________________________________________________________________    Amino Acid Type and Concentration: Effect on Haloperoxidase Killing of    Aspergillus fumigatus Spores                         CFU (Dicarboxylic Amino Acids & Amides)            Glucose                 (Amino Acid)                         l-Aspartic   l-Glutamic     CFU (Imino Acids)    Haloperoxidase            Oxidase                 μmol/ml (mM)                         Acid  l-Asparagine                                      Acid    l-Glutamine                                                     l-Proline                                                          l-Hydroxyproline    __________________________________________________________________________    0       0    0       520,000                               520,000                                      920,000 920,000                                                     260,000                                                          860,000    0       0    5       540,000                               540,000                                      260,000 420,000                                                     420,000                                                          640,000    0       0    0.5     460,000                               180,000                                      320,000 660,000                                                     300,000                                                          800,000    0       0    0.05    200,000                               240,000                                      580,000 300,000                                                     480,000                                                          740,000    0       0.6 Units                 0       340,000                               340,000                                      740,000 740,000                                                     420,000                                                          740,000    0       0.6 Units                 5       420,000                               340,000                                      280,000 400,000                                                     240,000                                                          540,000    0       0.6 Units                 0.5     360,000                               460,000                                      340,000 360,000                                                     360,000                                                          460,000    0       0.6 Units                 0.05    380,000                               160,000                                      640,000 560,000                                                     200,000                                                          720,000    20 pmol MPO            0.6 Units                 0       700,000                               700,000                                      1,640,000                                              1,640,000                                                     300,000                                                          940,000    20 pmol MPO            0.6 Units                 5       640,000                               660,000                                      840,000 1,080,000                                                     540,000                                                          1,000,000    20 pmol MPO            0.6 Units                 0.5     960,000                               340,000                                      820,000 1,000,000                                                     380,000                                                          900,000    20 pmol MPO            0.6 Units                 0.05    800,000                               680,000                                      820,000 750,000                                                     280,000                                                          680,000    20 pmol EPO            0.6 Units                 0       920,000                               920,000                                      1,060,000                                              1,060,000                                                     200,000                                                          860,000    20 pmol EPO            0.6 Units                 5       920,000                               1,340,000                                      1,100,000                                              820,000                                                     280,000                                                          840,000    20 pmol EPO            0.6 Units                 0.5     1,460,000                               440,000                                      540,000 660,000                                                     460,000                                                          1,260,000    20 pmol EPO            0.6 Units                 0.05    620,000                               1,260,000                                      900,000 400,000                                                     380,000                                                          680,000    __________________________________________________________________________

As shown in Table 3, none of the dicarboxylic amino acids or imino acidstested exhibited a significant haloperoxidase antimicrobial activityenhancing effect at any of the concentrations tested.

The hydroxyamino acids l-serine and l-threonine, and the basic aminoacids l-lysine, l-histidine and l-arginine were tested as describedabove. The results are shown in the following Table 4:

                                      TABLE 4    __________________________________________________________________________    Amino Acid Type and Concentration: Effect on Haloperoxidase Killing of    Aspergillus fumigatus Spores            Glucose                 (Amino Acid)                         CFU (Hydroxyamino Acids)                                       CFU (Basic Amino Acids)    Haloperoxidase            Oxidase                 μmol/ml (mM)                         l-Serine                               l-Threonine                                       l-Lysine                                            l-Histidine                                                  l-Arginine    __________________________________________________________________________    0       0    0       800,000                               760,000 520,000                                            700,000                                                  800,000    0       0    5       820,000                               520,000 520,000                                            440,000                                                  780.000    0       0    0.5     540,000                               800,000 460,000                                            840,000                                                  740,000    0       0    0.05    580,000                               660,000 460,000                                            840,000                                                  620,000    0       0.6 Units                 0       1,140,000                               400,000 340,000                                            400,000                                                  1,140,000    0       0.6 Units                 5       480,000                               800,000 240,000                                            520,000                                                  580,000    0       0.6 Units                 0.5     620,000                               360,000 480,000                                            740,000                                                  960,000    0       0.6 Units                 0.05    640,000                               560,000 520,000                                            560,000                                                  1,020,000    20 pmol MPO            0,6 Units                 0       700,000                               500,000 700,000                                            500,000                                                  700,000    20 pmol MPO            0.6 Units                 5       700,000                               400,000 380,000                                            720,000                                                  960,000    20 pmol MPO            0.6 Units                 0.5     660,000                               0       0    640,000                                                  740,000    20 pmol MPO            0.6 Units                 0.05    560,000                               12,000  0    520,000                                                  840,000    20 pmol EPO            0.6 Units                 0       840,000                               780,000 920,000                                            780,000                                                  840,000    20 pmol EPO            0.6 Units                 5       0     1,000,000                                       560,000                                            740,000                                                  960,000    20 pmol EPO            0.6 Units                 0.5     34,000                               0       620,000                                            600,000                                                  700,000    20 pmol EPO            0.6 Units                 0.05    860,000                               40,000  920,000                                            102,000                                                  900,000    __________________________________________________________________________

As shown in Table 4, the hydroxyamino acids l-serine and l-threonineboth significantly enhanced eosinoperoxidase killing of A. fumigatusspores while l-threonine and the basic amino acid l-lysine wereeffective in enhancing myeloperoxidase antimicrobial activity. The basicamino acids l-histidine and l-arginine exhibited no significantantimicrobial effects. Histidine is very reactive with singlet molecularoxygen, and as such, it would be expected to produce potent competitiveinhibition of haloperoxidase action which might mask its capacity tostimulate spore germination.

The sulfur amino acids l-cysteine and l-methionine, and the aromaticamino acids l-phenylalanine, l-tyrosine and l-tryptophan were tested asdescribed above. The results are shown in the following Table 5:

                                      TABLE 5    __________________________________________________________________________    Amino Acid Type and Concentration: Effect on Haloperoxidase Killing of    Aspergillus fumigatus Spores            Glucose                 (Amino Acid)                         CFU (Sulfur Amino Acids)                                       CFU (Aromatic Amino Acids)    Haloperoxidase            Oxidase                 μmol/ml (mM)                         l-Cysteine                                l-Methionine                                       l-Phenylalanine                                               l-Tyrosine                                                     l-Tryptophan    __________________________________________________________________________    0       0    0       380,000                                380,000                                       260,000 700,000                                                     380,000    0       0    5       540,000                                560,000                                       360,000 580,000                                                     800,000    0       0    0.5     500,000                                540,000                                       340,000 800,000                                                     640,000    0       0    0.05    380,000                                320,000                                       380,000 560,000                                                     700,000    0       0.6 Units                 0       460,000                                460,000                                       420,000 400,000                                                     460,000    0       0.6 Units                 5       580,000                                660,000                                       400,000 840,000                                                     920,000    0       0.6 Units                 0.5     420,000                                480,000                                       460,000 700,000                                                     580,000    0       0.6 Units                 0.05    360,000                                460,000                                       440,000 560,000                                                     280,000    20 pmol MPO            0.6 Units                 0       580,000                                580,000                                       300,000 500,000                                                     580,000    20 pmol MPO            0.6 Units                 5       580,000                                400,000                                       8,000   640,000                                                     700,000    20 pmol MPO            0.6 Units                 0.5     540,000                                480,000                                       4,000   460,000                                                     580,000    20 pmol MPO            0.6 Units                 0.05    720,000                                560,000                                       2,000   20,000                                                     1,040,000    20 pmol EPO            0.6 Units                 0       480,000                                480,000                                       200,000 780,000                                                     480,000    20 pmol EPO            0.6 Units                 5       680,000                                580,000                                       0       640,000                                                     860,000    20 pmol EPO            0.6 Units                 0.5     800,000                                560,000                                       0       920,000                                                     740,000    20 pmol EPO            0.6 Units                 0.05    240,000                                600,000                                       0       0     580,000    __________________________________________________________________________

As shown in Table 5, the sulfur amino acids were ineffective inenhancing antimicrobial activity of either eosinophil peroxidase ormyeloperoxidase. The aromatic amino acids l-phenylalanine and l-tyrosineboth exhibited significant enhancement of eosinophil peroxidase andmyeloperoxidase killing of A. fumigatus, while l-tryptophan exhibited nosignificant effect. These sulfur and aromatic amino acids are alsorelatively reactive with singlet molecular oxygen, and may competitivelyinhibit haloperoxidase action which would mask their capacity tostimulate spore germination. This might explain why l-phenylalanine andl-tyrosine are most effective when tested at a low concentration.

The effect of enantiomeric configuration of alanine and of isomericconfiguration and derivatisation of alanine were tested as describedabove. The results are shown in the following Table 6:

                                      TABLE 6    __________________________________________________________________________    Amino Acid Type and Concentration: Effect on Haloperoxidase Killing of    Aspergillus fumigatus Spores            Glucose                 (Amino Acid)                         CFU (Alinine Isomers and Derivatives)    Haloperoxidase            Oxidase                 μmol/ml (mM)                         l-Alanine                              d-Alanine                                    β-Alanine                                          l-Ala Methyl Ester                                                    l-Ala-l-Ala    __________________________________________________________________________    0       0    0       800,000                              740,000                                    740,000                                          740,000   740,000    0       0    5       520,000                              720,000                                    580,000                                          500,000   660,000    0       0    0.5     660,000                              760,000                                    920,000                                          540,000   640,000    0       0    0.05    520,000                              760,000                                    740,000                                          540,000   620,000    0       0.6 Units                 0       1,140,000                              760,000                                    760,000                                          760,000   760,000    0       0.6 Units                 5       980,000                              660,000                                    900,000                                          860,000   780,000    0       0.6 Units                 0.5     780,000                              700,000                                    620,000                                          740,000   680,000    0       0.6 Units                 0.05    760,000                              600,000                                    860,000                                          1,200,000 360,000    20 pmol MPO            0.6 Units                 0       700,000                              820,000                                    820,000                                          820,000   820,000    20 pmol MPO            0.6 Units                 5       0    14,000                                    760,000                                          0         660,000    20 pmol MPO            0.6 Units                 0.5     0    0     900,000                                          0         500,000    20 pmol MPO            0.6 Units                 0.05    4,000                              10,000                                    1,116,000                                          440,000   480,000    20 pmol EPO            0.6 Units                 0       840,000                              1,020,000                                    1,020,000                                          1,020,000 1,020,000    20 pmol EPO            0.6 Units                 5       0    0     940,000                                          0         660,000    20 pmol EPO            0.6 Units                 0.5     0    0     880,000                                          0         1,360,000    20 pmol EPO            0.6 Units                 0.05    10,000                              10,000                                    720,000                                          580,000   680,000    __________________________________________________________________________

As shown in Table 6, both the l- and d-enantiomers of alanine werehighly effective in enhancing the myeloperoxidase and eosinophilperoxidase killing of A. fumigatus, while β-alanine exhibited nosignificant enhancing effect. Similarly, the methyl ester of l-alanineproduced significant enhancement of antimicrobial activity. Thel-alanine-l-alanine dipeptide exhibited no significant enhancementactivity.

The effect of enantiomeric configuration of threonine was also tested asdescribed above. The results are shown in the following Table 7:

                  TABLE 7    ______________________________________    Amino Acid Type and Concentration: Effect on Haloperoxidase    Killing of Aspergillus fumigatus Spores                   (Amino                   Acid)    CFU (Enantiomers of            Glucose                   μmol/ml                            Hydroxyamino Acids)    Haloperoxidase              Oxidase  (mM)     l-Threonine                                        d-Threonine    ______________________________________    0         0        0        760,000 800,000    0         0        5        520,000 820,000    0         0        0.5      800,000 760,000    0         0        0.05     660,000 680,000    0         0.6 Units                       0        400,000 1,140,000    0         0.6 Units                       5        800,000 460,000    0         0.6 Units                       0.5      360,000 720,000    0         0.6 Units                       0.05     560,000 720,000    20 pmol MPO              0.6 Units                       0        500,000 700,000    20 pmol MPO              0.6 Units                       5        400,000 420,000    20 pmol MPO              0.6 Units                       0.5      0       0    20 pmol MPO              0.6 Units                       0.05     12,000  280,000    20 pmol EPO              0.6 Units                       0        780,000 840,000    20 pmol EPO              0.6 Units                       5        1,000,000                                        0    20 pmol EPO              0.6 Units                       0.5      0       0    20 pmol EPO              0.6 Units                       0.05     40,000  520,000    ______________________________________

As shown in Table 7, both the l- and d-enantiomers of threoninesignificantly enhanced the myeloperoxidase and eosinophil peroxidasekilling of A. fumigatus.

The effect of using the α-keto acid forms of enhancing α-amino acids wastested with l-alanine and pyruvic acid, and with glycine and glyoxylicacid, as described above. The results are shown in the following Table8:

                                      TABLE 8    __________________________________________________________________________    Amino Acid Type and Concentration:    Effect on Haloperoxidase Killing of Aspergillus fumigatus Spores            Glucose                 (Amino Acid)                         CFU (Amino Acid versus Alpha Keto Acid)    Haloperoxidase            Oxidase                 μmol/ml (mM)                         l-Alanine                              Pyruvic Acid                                     Glycine                                          Glyoxylic Acid    __________________________________________________________________________    0       0    0       800,000                              860,000                                     860,000                                          860,000    0       0    5       520,000                              880,000                                     720,000                                          600,000    0       0    0.5     660,000                              700,000                                     480,000                                          700,000    0       0    0.05    520,000                              660,000                                     640,000                                          560,000    0       0.6 Units                 0       1,140,000                              740,000                                     740,000                                          740,000    0       0.6 Units                 5       980,000                              760,000                                     520,000                                          560,000    0       0.6 Units                 0.5     780,000                              600,000                                     760,000                                          600,000    0       0.6 Units                 0.05    760,000                              480,000                                     800,000                                          660,000    20 pmol MPO            0.6 Units                 0       700,000                              940,000                                     940,000                                          940,000    20 pmol MPO            0.6 Units                 5       0    820,000                                     0    1,060,000    20 pmol MPO            0.6 Units                 0.5     0    880,000                                     0    660,000    20 pmol MPO            0.6 Units                 0.05    4,000                              580,000                                     90,000                                          580,000    20 pmol EPO            0.6 Units                 0       840,000                              860,000                                     860,000                                          660,000    20 pmol EPO            0.6 Units                 5       0    640,000                                     0    740,000    20 pmol EPO            0.6 Units                 0.5     0    560,000                                     0    720,000    20 pmol EPO            0.6 Units                 0.05    10,000                              780,000                                     460,000                                          740,000    __________________________________________________________________________

As shown in Table 8, pyruvic acid and glyoxylic acid do not exhibit theactivity enhancing effect of l-alanine and glycine.

Example 3 Effect of Antimicrobial Activity Enhancing Agents on OtherAntifungal Systems

In order to determine the effect of l-alanine on the antimicrobialeffect of the common antifungal compounds nystatin and amphotericin B,the procedure of Example 2 was followed using nystatin or amphotericin Bin place of the haloperoxidase-glucose oxidase of Example 2 and 0(control) or 10 μmol/ml of l-alanine with Fusarium moniliforme as themicrobe. The results are shown in the following table 9:

                  TABLE 9    ______________________________________    Effect of l-Alanine on Nystatin and    Amphotericin B Antifungal Activity    Antifungal Agent                    l-Alanine CFU (F. moniliforme    Final Concentration                    μmol/test                              ATCC 38159)    ______________________________________    None            None      940,000    Nystatin, 400 μg/ml                    None      120,000    Nystatin, 40 μg/ml                    None      340,000    Nystatin, 4 μg/ml                    None      500,000    None            10        1,020,000    Nystatin, 400 μg/ml                    10         28,000    Nystatin, 40 μg/ml                    10        124,000    Nystatin, 4 μg/ml                    10        220,000    None            None      880,000    Amphotericin B, 250 μg/ml                    None      320,000    Amphotericin B, 25 μg/ml                    None      500,000    Amphotericin B, 2.5 μg/ml                    None      880,000    None            10        840,000    Amphotericin B, 250 μg/ml                    10        340,000    Amphotericin B, 25 μg/ml                    10        460,000    Amphotericin B, 2.5 μg/ml                    10        800,000    ______________________________________

As can be seen in Table 9, the addition of l-alanine doubled thenystatin-dependent killing of Fusarium moniliforme but had no effect onamphotericin B-dependent killing of Fusarium moniliforme.

The foregoing procedure was repeated using the antiseptic compoundshydrogen peroxide (H₂ O₂) and sodium hypochlorite (NaClO) with spores ofAspergillus fumigatus and Fusarium moniliforme. The results are shown inthe following Table 10:

                  TABLE 10    ______________________________________    Effect of l-Alanine on Hydrogen Peroxide and Sodium    Hypochlorite Antifungal Activity                 l-Alanine                          CFU        CFU    Antifungal Agent                 μmol/ (A. fumigatus                                     (F. moniliforme    Final Concentration                 test     ATCC 10894)                                     ATCC 38159)    ______________________________________    None         None     360,000    460,000    H.sub.2 O.sub.2, 3 mg/ml                 None     580,000        0    H.sub.2 O.sub.2, 0.3 mg/ml                 None     400,000    140,000    H.sub.2 O.sub.2, 0.03 mg/ml                 None     440,000    400,000    H.sub.2 O.sub.2, 0.003 mg/ml                 None     640,000    500,000    None         10       700,000    580,000    H.sub.2 O.sub.2, 3 mg/ml                 10       720,000        0    H.sub.2 O.sub.2, 0.3 mg/ml                 10       280,000    340,000    H.sub.2 O.sub.2, 0.03 mg/ml                 10       660,000    300,000    H.sub.2 O.sub.2, 0.003 mg/ml                 10       500,000    310,000    None         None     300,000    284,000    NaClO, 1 mg/ml                 None      10,000     50,000    NaClO, 0.1 mg/ml                 None      26,000    298,000    NaClO 0.01 mg/ml                 None     560,000    290,000    NaClO, 0.001 mg/ml                 None     640,000    294,000    None         10       700,000    380,000    NaClO, 1 mg/ml                 10        26,000    292,000    NaClO, 0.1 mg/ml                 10        54,000    404,000    NaClO, 0.01 mg/ml                 10       580,000    404,000    NaClO, 0.001 mg/ml                 10       280,000    304,000    ______________________________________

As with amphotericin B, no significant enhancement of hydrogen peroxideor hypochlorite antisepsis is seen when used in combination withl-alanine. In fact, l-alanine appears to inhibit peroxide and especiallyhypochlorite killing of the fungi. Under such conditions l-alanineprobably acts as a competitive inhibitor.

Example 4 Effect of l-Alanine on Haloperoxidase Killing of AdditionalOrganisms

The effect of l-alanine on the halperoxidase antimicrobial activityagainst Fusarium moniliforme, Tricophyton rubrum and Crytococcusneoformans was determined following the procedure of Example 2 using 0(control) or 10 μmol/ml of l-alanine and 2, 10 or 50 pmol ofmyeloperoxidase or eosinophil peroxidase per test against theseorganisms. The results are shown in the following Table 11 (F.moniliforme, ATCC #38159), Table 12 (T. rubrum, ATCC #28188, 18753 and18758) and Table 13 (C. neoformans, ATCC #14115):

                  TABLE 11    ______________________________________    Haloperoxidase Killing of Fusarium moniliforme              Glucose   l-Alanine Fusarium moniliforme    Haloperoxidase              Oxidase   μmol/test                                  ATCC #38159    ______________________________________    None      None      10        1,720,000    None      0.6 Units None      1,380,000    None      0.6 Units 10        1,760,000    50 pmol MPO              None      10        1,640,000    50 pmol MPO              0.6 Units None      0    10 pmol MPO              0.6 Units None      0    50 pmol MPO              0.6 Units 10        0    10 pmol MPO              0.6 Units 10        8,000    50 pmol EPO              None      10        8,000    50 pmol EPO              0.6 Units None      0    10 pmol EPO              0.6 Units None      0     2 pmol EPO              0.6 Units None      0    50 pmol EPO              0.6 Units 10        0    10 pmol EPO              0.6 Units 10        0     2 pmol EPO              0.6 Units 10        0    ______________________________________

                                      TABLE 12    __________________________________________________________________________    Haloperoxidase Killing of Trichophyton                     l-Alanine                           Trichophyton rubrum                                     Trichophyton rubrum                                               Trichophyton rubrum    Haloperoxidase            Glucose Oxidase                     μmol/test                           ATCC #28188                                     ATCC #18753                                               ATCC #18758    __________________________________________________________________________    None    None     10    1,700,000 200,000   1,440,000    None    0.6 Units                     10    1,580,000 276,000   128,000    50 pmol MPO            None     10    2,680,000 192,000   720,000    50 pmol MPO            0.6 Units                     None  1,240,000 132,000   414,000    50 pmol MPO            0.6 Units                     10    84,000    18,000    0    10 pmol MPO            0.6 Units                     10    184,000   62,000    0     2 pmol MPO            0.6 Units                     10    1,380,000 310,000   4,000    50 pmol EPO            None     10    3,600,000 332,000   1,800,000    50 pmol EPO            0.6 Units                     None  1,180,000 22,000    244,000    50 pmol EPO            0.6 Units                     10    0         0         0    10 pmol EPO            0.6 Units                     10    0         0         0     2 pmol EPO            0.6 Units                     10    0         0         0    __________________________________________________________________________

                  TABLE 13    ______________________________________    Haloperoxidase Killing of Cryptococcus neoformans                                   CFU (Cryptococcus              Glucose    l-Alanine neoformans)    Haloperoxidase              Oxidase    μmol/test                                   ATCC #14115)    ______________________________________    None      None       10        960,000    None      0.6 Units  None      680,000    None      0.6 Units  10        480,000    50 pmol MPO              None       10        480,000    50 pmol MPO              0.6 Units  None      480,000    50 pmol MPO              0.6 Units  10        440,000    10 pmol MPO              0.6 Units  10        860,000     2 pmol MPO              0.6 Units  10        220,000    50 pmol EPO              None       10        660,000    50 pmol EPO              0.6 Units  None      460,000    50 pmol EPO              0.6 Units  10        0    10 pmol EPO              0.6 Units  10        0     2 pmol EPO              0.6 Units  10        640,000    ______________________________________

As shown in Table 11, myeloperoxidase and eosinophil peroxidase are bothhighly effective against Fusarium moniliforme either in the presence orabsence of l-alanine. In fact, EPO was found to be effective in theabsence of glucose oxidase. As shown in Table 12, complete killing ofTrichophyton is obtained with eosinophil peroxidase in the presence ofl-alanine, while a significant enhancement of myeloperoxidase killing isobtained.

As shown in Table 13, l-alanine also significantly enhances eosinophilperoxidase killing of Crytococcus neoformans, while some enhancement inmyeloperoxidase activity is seen.

Example 5 Effect of Cholesterol Oxidase Concentration onOxidase-Haloperoxidase Microbicidal Action Against Candida albicans

The effect of cholesterol oxidase concentration onoxidase-haloperoxidase microbicidal action against Candida albicans wasdetermined by following the procedure of Example 1, except that eachtest contained a different quantity of cholesterol oxidase from Nocardiaerythropolis (0.1 unit=4 μg) as indicated, 10 pmol (1.4 μg) porcine MPO(ExOxEmis, Inc., San Antonio, Texas, U.S.A., Lot#1899201) or 10 pmol(0.7 μg) porcine EPO (ExOxEmis, Inc., San Antonio, Tex., U.S.A.,Lot#1929201) in 50 mM Acetate Buffer containing 100 mEq/L Cl⁻, 1 mEq/LBr⁻, and 1 mM l-alanine. The pH was 6.7 with 50 mM MOPS as buffer. Thefinal suspension contained 7 mM (7 μmol/ml) cholesterol in 8.5% ethanol.The final volume was 1 ml. After two hours incubation at 37° C., themicrobes were plate on Sabouraud's dextrose agar. The results areexpressed in Table 14 as the colony forming units (CFU) counted.

                  TABLE 14    ______________________________________    Effect of Cholesterol Oxidase Concentration on    Oxidase-Haloperoxidase Microbicidal Action    Against Candida albicans:               Cholesterol    Organism   Oxidase      Haloperoxidase                                        CFU    ______________________________________    Cand. albicans               None         None        400,000    Cand. albicans                0.1 Unit    None        480,000    Cand. albicans                0.1 Unit †                            10 pmol MPO 420,000    Cand. albicans                0.1 Unit    10 pmol MPO 0    Cand. albicans                0.05 Unit   10 pmol MPO 0    Cand. albicans               0.025 Unit   10 pmol MPO 0    Cand. albicans               0.013 Unit   10 pmol MPO 76,000    Cand. albicans               None         10 pmol MPO 560,000    Cand. albicans                0.1 Unit †                            10 pmol EPO 400,000    Cand. albicans                0.1 Unit    10 pmol EPO 0    Cand. albicans                0.05 Unit   10 pmol EPO 0    Cand. albicans               0.025 Unit   10 pmol EPO 200    Cand. albicans               0.013 Unit   10 pmol EPO 180,000    Cand. albicans               None         10 pmol EPO 360,000    ______________________________________

As shown in Table 14, complete killing of C. albicans was observed with0.025 unit cholesterol oxidase plus 10 pmol MPO and 1 mM l-alanine. Inthe absence of l-alanine, no killing was observed with 0.1 unitcholesterol oxidase with 10 pmol MPO. Similar results were obtained withEPO was substituted as the haloperoxidase.

Example 6 Effect of Cholesterol Oxidase Concentration onOxidase-Haloperoxidase Microbicidal Action Against Bacteria

The procedure of Example 5 was followed using Escherichia coli in placeof the C. albicans of Example 5. Each test contained the indicatedquantity of cholesterol oxidase from Nocardia erythropolis (0.1 unit=4μg), 10 pmol (1.4 μg) porcine MPO (ExOxEmis, Inc. San Antonio, Tex.,U.S.A., Lot#1899201) or 10 pmol (0.7 μg) porcine EPO (ExOxEmis, Inc.,San Antonio, Tex., U.S.A., Lot#1929201) in 50 mM Acetate Buffercontaining 100 mEq/L Cl⁻, 1 mEq/L Br⁻, and 1 mM l-alanine. The pH was6.7 with 50 mM MOPS as buffer. The final suspension contained 7 mM (7μmol/ml) cholesterol in 8.5% ethanol. The final volume was 1 ml. Aftertwo hours incubation at 37° C., the microbes were plated on trypticasesoy agar. The results are expressed in the following Table 15 as thecolony forming units (CFU) counted.

                  TABLE 15    ______________________________________    Effect of Cholesterol Oxidase Concentration on    Oxidase-Haloperoxidase Microbicidal Action    Against Escherichia coli:              Cholesterol    Organism  Oxidase     Haloperoxidase                                      CFU    ______________________________________    Escherichia coli              None        None        13,500,000    Escherichia coli               0.1 Unit   None        2,300,000    Escherichia coli               0.1 Unit †                          10 pmol MPO 0    Escherichia coli               0.1 Unit   10 pmol MPO 0    Escherichia coli               0.05 Unit  10 pmol MPO 0    Escherichia coli              0.025 Unit  10 pmol MPO 0    Escherichia coli              0.013 Unit  10 pmol MPO    Escherichia coli              None        10 pmol MPO 14,000,000    Escherichia coli               0.1 Unit †                          10 pmol EPO 0    Escherichia coli               0.1 Unit   10 pmol EPO 0    Escherichia coli               0.05 Unit  10 pmol EPO 0    Escherichia coli              0.025 Unit  10 pmol EPO 0    Escherichia coli              0.013 Unit  10 pmol EPO 0    Escherichia coli              None        10 pmol EPO 8,200,000    ______________________________________     † indicates that the 50 mM Acetate Buffer did NOT contain lalanine

As shown in Table 15, complete MPO and EPO killing of Escherichia coliwas observed at all cholesterol oxidase concentrations tested (0.0125 to0.1 unit) in the absence or presence of 1 mM l-alanine. The same resultswere observed with Staphylococcus aureus (data not shown).

Example 7 Oxidase-Haloperoxidase Killing of Bacterial, Yeast and FungalSpores Using Choline Oxidase

The procedure of Example 1 was followed except that 0.2 unit cholineoxidase was employed as the H₂ O₂ -generating oxidase. Where indicatedthe reaction contained 0.2 unit (i.e., 20 μg) choline oxidase fromAlcaligenes sp., 20 pmol (2.8 μg) porcine MPO (ExOxEmis, Inc., SanAntonio, Tex., U.S.A., Lot#1899201) or 20 pmol (1.5 μg) porcine EPO(ExOxEmis, Inc., San Antonio, Tex., U.S.A., Lot#1929201) in 50 mMAcetate Buffer containing 100 mEq/L Cl⁻, and 1 mEq/L Br⁻. The pH was 7with 50 mM MOPS as buffer. The final concentration of choline was 150 mM(150 μmol/ml). The final volume was 1 ml. After four hours incubation at22° C. the microbes were plated (S. aureus was plated on trypticase soyagar; C. albicans and A. fumigatus were plated on Sabouraud's dextroseagar). The results are expressed in Table 16 as the colony forming units(CFU) counted.

                  TABLE 16    ______________________________________    Effect of l-Alanine on Choline Oxidase-Haloperoxidase    Killing of Staphylococcus aureus, Candida albicans,    and Aspergillus fumigatus Spores:               Choline    Organism   Oxidase    Haloperoxidase                                      CFU    ______________________________________    Staph. aureus               None       None        19,400,000    Staph. aureus               0.2 Unit   None        13,800,000    Staph. aureus               0.2 Unit †                          None        15,400,000    Staph. aureus               0.2 Unit   20 pmol MPO 0    Staph. aureus               0.2 Unit †                          20 pmol MPO 0    Staph. aureus               0.2 Unit   20 pmol EPO 0    Staph. aureus               0.2 Unit †                          20 pmol EPO 0    Cand. albicans               None       None        1,460,000    Cand. albicans               0.2 Unit   None        1,200,000    Cand. albicans               0.2 Unit †                          None        1,180,000    Cand. albicans               0.2 Unit   20 pmol MPO 1,120,000    Cand. albicans               0.2 Unit †                          20 pmol MPO 0    Cand. albicans               0.2 Unit   10 pmol EPO 640,000    Cand. albicans               0.2 Unit †                          20 pmol EPO 0    Asperg. fumigatus               None       None        1,260,000    Asperg. fumigatus               0.2 Unit   None        1,260,000    Asperg. fumigatus               0.2 Unit †                          None        1,300,000    Asperg. fumigatus               0.2 Unit   20 pmol MPO 800,000    Asperg. fumigatus               0.2 Unit †                          20 pmol MPO 0    Asperg. fumigatus               0.2 Unit   20 pmol EPO 740,000    Asperg. fumigatus               0.2 Unit †                          20 pmol EPO 0    ______________________________________     †indicates that the 50 mM Acetate Buffer contained 1 mM lalanine.

Example 8 Oxidase-Haloperoxidase Killing of Bacterial, Yeast and FungalSpores Using Lactate Oxidase

The procedure of Example 7 was followed except that 0.2 unit lactateoxidase was employed as the H₂ O₂ -generating oxidase. Where indicatedthe reaction contained 0.2 unit (i.e., 5 μg) lactate oxidase fromPediococcus sp., 20 pmol (2.8 μg) porcine MPO (ExOxEmis, Inc., SanAntonio, Tex., U.S.A., Lot#1899201) or 20 pmol (1.5 μg) porcine EPO(ExOxEmis, Inc., San Antonio, Tex., U.S.A., Lot#1929201) in 50 mMAcetate Buffer containing 100 mEq/L Cl⁻, and 1 mEq/L Br⁻. The pH was 7with 50 mM MOPS as buffer. The final concentration of lactate was 150 mM(150 μmol/ml). The final volume was 1 ml. After four hours incubation(22° C.) the microbes were plated. S. aureus was plated on trypticasesoy agar. C. albicans and A. fumigatus were plated on Sabouraud'sdextrose agar. The results are expressed in Table 17 as the colonyforming units (CFU) counted.

                  TABLE 17    ______________________________________    Effect of l-Alanine on Lactate Oxidase-Haloperoxidase    Killing of Staphylococcus aureus, Candida albicans,    and Aspergillus fumigatus Spores:                Lactate    Organism    Oxidase    Haloperoxidase                                       CFU    ______________________________________    Staph. aureus                None       None        19,400,000    Staph. aureus                0.2 Unit   None        23,400,000    Staph. aureus                0.2 Unit †                           None        24,400,000    Staph. aureus                0.2 Unit   20 pmol MPO 0    Staph. aureus                0.2 Unit †                           20 pmol MPO 0    Staph. aureus                0.2 Unit   20 pmol EPO 0    Staph. aureus                0.2 Unit †                           20 pmol EPO 0    Cand. albicans                None       None        1,460,000    Cand. albicans                0.2 Unit   None        1,480,000    Cand. albicans                0.2 Unit †                           None        1,020,000    Cand. albicans                0.2 Unit   20 pmol MPO 1,380,000    Cand. albicans                0.2 Unit †                           20 pmol MPO 1,500,000    Cand. albicans                0.2 Unit   20 pmol EPO 1,400,000    Cand. albicans                0.2 Unit †                           20 pmol EPO 1,180,000    Asperg. fumigatus                None       None        1,260,000    Asperg. fumigatus                0.2 Unit   None        860,000    Asperg. fumigatus                0.2 Unit †                           None        760,000    Asperg. fumigatus                0.2 Unit   20 pmol MPO 740,000    Asperg. fumigatus                0.2 Unit †                           20 pmol MPO 400,000    Asperg. fumigatus                0.2 Unit   20 pmol EPO 760,000    Asperg. fumigatus                0.2 Unit †                           20 pmol EPO 18,000    ______________________________________     †indicates that the 50 mM Acetate Buffer contained 1 mM lalanine.

Example 9 Oxidase-Haloperoxidase Killing of Bacterial, Yeast and FungalSpores Using Alcohol Oxidase

The procedure of Example 7 was followed except that 0.2 unit alcoholoxidase was employed as the H₂ O₂ -generating oxidase. Where indicatedthe reaction contained 0.2 unit (i.e., 21 μg) alcohol oxidase fromCandida boidinii, 20 pmol (2.8 μg) porcine MPO (ExOxEmis, Inc., SanAntonio, Tex., U.S.A., Lot#1899201) or 20 pmol (1.5 μg) porcine EPO(ExOxEmis, Inc., San Antonio, Tex., U.S.A., Lot#1929201) in 50 mMAcetate Buffer containing 100 mEq/L Cl⁻, and 1 mEq/L Br⁻. The pH was 7with 50 mM MOPS as buffer. The final concentration of alcohol was 150 mM(150 μmol/ml). The final volume was 1 ml. After four hours incubation at22° C. the microbes were plated. S. aureus was plated on trypticase soyagar. C. albicans and A. fumigatus were plated on Sabouraud's dextroseagar. The results are expressed in Table 18 as the colony forming units(CFU) counted.

                  TABLE 18    ______________________________________    Effect of l-Alanine on Alcohol Oxidase-Haloperoxidase    Killing of Staphylococcus aureus, Candida albicans,    and Aspergillus fumigatus Spores:                Alcohol    Organism    Oxidase    Haloperoxidase                                       CFU    ______________________________________    Staph. Aureus                None       None        19,400,000    Staph. Aureus                0.2 Unit   None        21,200,000    Staph. Aureus                0.2 Unit †                           None        19,400,000    Staph. Aureus                0.2 Unit   20 pmol MPO 840,000    Staph. Aureus                0.2 Unit †                           20 pmol MPO 760,000    Staph. Aureus                0.2 Unit   20 pmol EPO 0    Staph. Aureus                0.2 Unit †                           10 pmol EPO 0    Cand. Albicans                None       None        1,460,000    Cand. Albicans                0.2 Unit   None        1,100,000    Cand. Albicans                0.2 Unit †                           None        1,460,000    Cand. Albicans                0.2 Unit   20 pmol MPO 1,180,000    Cand. Albicans                0.2 Unit †                           20 pmol MPO 1,280,000    Cand. Albicans                0.2 Unit   20 pmol EPO 1,220,000    Cand. Albicans                0.2 Unit †                           20 pmol EPO 1,160,000    Asperg. fumigatus                None       None        1,260,000    Asperg. fumigatus                0.2 Unit   None        620,000    Asperg. fumigatus                0.2 Unit †                           None        700,000    Asperg. fumigatus                0.2 Unit   20 pmol MPO 1,560,000    Asperg. fumigatus                0.2 Unit †                           20 pmol MPO 320,000    Asperg. fumigatus                0.2 Unit   20 pmol EPO 1,040,000    Asperg. fumigatus                0.2 Unit †                           20 pmol EPO 16,000    ______________________________________     †indicates that the 50 mM Acetate Buffer contained 1 mM lalanine.

Cholesterol oxidase (Table 1), choline oxidase (Table 16), and lactateoxidase (Table 17) in combination with either MPO or EPO producedcomplete killing of Staphylococcus aureus in the presence or absence ofl-alanine. With alcohol oxidase (Table 18), killing was complete withEPO, but only partial with MPO either with or without l-alanine.

Candida albicans was totally killed by cholesterol oxidase (Table 1) andcholine oxidase (Table 16) plus MPO or EPO only in the presence ofl-alanine. In the absence of l-alanine, microbicidal action was limitedto approximately fifty percent kill. Lactate oxidase (Table 17) andalcohol oxidase (Table 18) were not effective in driving MPO or EPOkilling of Candida albicans in the presence or absence of l-alanine.

In the presence of l-alanine, both cholesterol oxidase (Table 1) andcholine oxidase (Table 16) supported complete killing of Aspergillusfumigatus spores by MPO or EPO. However, only partial killing wasobserved in the absence of l-alanine. Although incomplete, both lactateoxidase (Table 17) and alcohol oxidase (Table 18) produced greater than90% spore kill with EPO and greater than 50% spore kill with MPO in thepresence of l-alanine. In the absence of l-alanine, neither lactateoxidase or alcohol oxidase supported MPO or EPO killing of Aspergillusfumigatus spores.

Example 10 The Effect of Potential Activator Substances on CholesterolOxidase-Haloperoxidase Killing of Bacillus and Aspergillus Spores.

The microbiology literature describes several substances that mightserve as activators of germination of spores and vegetative forms.Conidiospore germination has been reported to require glucose, phosphateand an amino acid. L-proline or l-alanine fulfill the amino acidrequirement. Other amino acids and vitamins are less effective(Yanigita, 1957, Arch Mikrobiol 26:329).

Several other organic compounds have been reported to stimulategermination. These include phenethyl alcohol (Lingappa et al., 1970,Arch Mikrobiol 72:97), coumarin (Weber and Hess, 1974, The Fungal Spore,p. 178, Wiley-Interscience), and furfural derivatives (Sussman et al.,1959, Mycologia 51:237).

The effect of l-alanine and l-proline on cholesteroloxidase-haloperoxidase killing of Bacillus cereus endospores contained0.1 unit (i.e., 4 μg) cholesterol oxidase from Nocardia erythropolis, 20pmol (2.8 μg) porcine MPO (ExOxEmis, Inc., San Antonio, Tex., U. S. A.,Lot#1899201) or 20 pmol (1.5 μg) porcine EPO (ExOxEmis, Inc., SanAntonio, Tex., U. S. A., Lot#1929201), and the indicated quantity ofl-alanine or l-proline in 50 mM Acetate Buffer containing 100 mEq/L Cl⁻,and 1 mEq/L Br⁻. The pH was adjusted to ˜7 by addition of 50 mM MOPSbuffer. The final concentration of cholesterol was 5 mM (5 μmol/ml) in8.5% ethanol. The final volume was 1 ml. After the indicated period ofincubation (22° C.), the surviving microbes were plated on trypticasesoy agar. The results are shown in Table 19 as the colony forming units(CFU) counted.

                                      TABLE 19    __________________________________________________________________________    Effects of l-Alanine and l-Proline on Cholesterol Oxidase-Haloperoxidase    Microbicidal Action Against Bacillus cereus Spores:    Treatment:         Bacillus cereus Survival,    Haloper- Activator CFU (Post Exposure Time in Hours)    oxidase  Substance 0.5 Hr.                           1 Hr.                               1.5 Hr.                                   2 Hrs.                                       3 Hrs.    __________________________________________________________________________    None     None      900,000                           460,000                               720,000                                   720,000                                       520,000    None     l-alanine, 10 μmol                       860,000                           660,000                               560,000                                   520,000                                       400,000    None     l-proline, 10 μmol                       820,000                           760,000                               620,000                                   320,000                                       540,000    MPO, 20 pmol             None      0   0   0   0   0    MPO, 20 pmol             l-alanine, 10 μmol                       0   0   0   0   0    MPO, 20 pmol             l-proline, 10 μmol                       0   0   0   0   0    EPO, 20 pmol             None      2,000                           2,000                               0   0   0    EPO, 20 pmol             l-alanine, 10 μmol                       2,000                           0   0   0   0    EPO, 20 pmol             l-proline, 10 μmol                       0   2,000                               0   0   0    __________________________________________________________________________

After thirty minutes of incubation, cholesterol oxidase (0.1 unit) withMPO (20 pmol) produced complete killing of Bacillus cereus spores witheither l-alanine or l-proline as the activator. However, killing wasalso complete without any added enhancer substance. Although a longerincubation period was required, similar results were obtained usingcholesterol oxidase with EPO. In the absence of haloperoxidase, additionof l-alanine or l-proline did not significantly increase killingactivity of cholesterol oxidase. The direct sensitivity of thesebacterial endospores to the oxidase-haloperoxidase killing is so greatthat any effect of l-alanine or l-proline is hidden in the total killobserved as early as thirty minutes.

The effect of l-alanine, l-proline, coumarin, and phenethyl alcohol oncholesterol oxidase-haloperoxidase killing of Aspergillus fumigatusspores was determined by following the foregoing procedure except thatthe reaction contained 0.1 unit (i.e., 4 μg) cholesterol oxidase fromNocardia erythropolis, 20 pmol (2.8 μg) porcine MPO (ExOxEmis, Inc., SanAntonio, Tex., U. S. A., Lot#1899201) or 20 pmol (1.5 μg) porcine EPO(ExOxEmis, Inc., San Antonio, Tex., U. S. A., Lot#1929201), and theindicated quantity of substance to be tested in 50 mM Acetate Buffercontaining 100 mEq/L Cl⁻, and 1 mEq/L Br⁻. The pH was adjusted to ˜7 byaddition of 50 mM MOPS buffer. The final concentration of cholesterolwas 5 mM (5 μmol/ml) in 8.5% ethanol. The final volume was 1 ml. Afterthe indicated period of incubation (37° C.), Aspergillus fumigatus wasplated on Sabouraud's dextrose agar. The results are expressed in Table20 as the colony forming units (CFU) counted. PEA is the acronym forphenethyl alcohol. Of the four potential activator substances tested,l-alanine alone increased the kill capacity of cholesterol oxidase witheither MPO or EPO. In the absence of haloperoxidase, none of theactivators increased the killing activity of cholesterol oxidase.

                                      TABLE 20    __________________________________________________________________________    Effects of l-Alanine, l-Proline, Coumarin, and Phenethyl Alcohol on    Cholesterol Oxidase-Haloperoxidase Microbicidal Action Against    Aspergillus fumigatus Spores:                     A. fumigatus Survival,    Treatment:            Activator                     CFU (Post Exposure Time in Hours)    Haloperoxidase            Substance                     1 Hr.                          1.5 Hr.                              2 Hrs.                                   3 Hrs.                                       4 Hrs.    __________________________________________________________________________    None    None     710,000                          760,000                              530,000                                   340,000                                       240,000    None    l-alanine, 10 μmol                     780,000                          720,000                              500,000                                   340,000                                       340,000    None    l-proline, 10 μmol                     900,000                          540,000                              560,000                                   400,000                                       200,000    None    coumarin, 1 μmol                     320,000                          700,000                              480,000                                   240,000                                       500,000    None    PEA, 5 μmol                     380,000                          600,000                              360,000                                   440,000                                       480,000    MPO, 20 pmol            None     520,000                          940,000                              500,000                                   410,000                                       550,000    MPO, 20 pmol            l-alanine, 10 μmol                     0    0   0    0   0    MPO, 20 pmol            l-proline, 10 μmol                     8360,000                          880,000                              760,000                                   700,000                                       260,000    MPO, 20 pmol            coumarin, 1 μmol                     740,000                          620,000                              500,000                                   620,000                                       660,000    MPO, 20 pmol            PEA, 5 μmol                     500,000                          600,000                              420,000                                   420,000                                       520,000    EPO, 20 pmol            None     920,000                          690,000                              440,000                                   710,000                                       760,000    EPO, 20 pmol            l-alanine, 10 μmol                     0    0   0    0   0    EPO, 20 pmol            l-proline, 10 μmol                     1,100,000                          840,000                              1,000,000                                   500,000                                       520,000    EPO, 20 pmol            coumarin, 1 μmol                     760,000                          780,000                              420,000                                   340,000                                       300,000    EPO, 20 pmol            PEA, 5 μmol                     780,000                          600,000                              480,000                                   460,000                                       320,000    __________________________________________________________________________

Incorporation of l-alanine into the formulation greatly increases thefungicidal action of oxidase-haloperoxidase against both spores andvegetative forms. L-alanine appears to provide the amine nitrogen, CO₂,acetyl-CoA, and reducing equivalents required for germination and growthand as such labilizes the fungi to the action of haloperoxidases. Aspreviously shown with haloperoxidase-glucose oxidase killing (Table 3),l-proline is not effective as an enhancer substance.

Example 11 Binary Antiseptic Consisting of (1) CholesterolOxidase-Haloperoxidase and (2) Cholesterol in Solid Form.

The cholesterol oxidase-haloperoxidase system was constructed as abinary formulation consisting of: (1) a solution containing cholesteroloxidase-haloperoxidase plus the antimicrobial activity enhancing agentl-alanine, and (2) cholesterol in the form of a solid wafer. Thecholesterol wafers were prepared by dissolving cholesterol in ether,pouring the cholesterol solution on a surface, and allowing the ether toevaporate. The resulting sheet of cholesterol was cut into appropriatelysized rectangles of approximately 10 mg.

Cholesterol wafers (approximately 10 mg/wafer) were placed in test tubescontaining Aspergillus fumigatus spores. Cholesterol oxidase with andwithout MPO or EPO was added to the tubes to initiate fungicidal action.The system was tested with and without l-alanine, and with and withoutethanol. Ethanol was added to test its capacity to facilitatecholesterol solubilization. Increased cholesterol solubility would meanincreased cholesterol available as substrate for the oxidase. Onceinitiated, the system was allowed to incubate at ambient temperature(22° C.) for time intervals ranging from thirty minutes to four hours.The reaction contained 0.1 units (i.e., 4 μg) cholesterol oxidase fromNocardia erythropolis, 10 pmol (1.4 μg) porcine MPO (ExOxEmis, Inc., SanAntonio, Tex., U. S. A., Lot#1899201) or 10 pmol (0.7 μg) porcine EPO(ExOxEmis, Inc., San Antonio, Tex. U. S. A., Lot#1929201) in 50 mMAcetate Buffer containing 100 mEq/L Cl⁻, and 1 mEq/L Br⁻. The pH wasadjusted to ˜7 by addition of 50 mM MOPS buffer. The substratecholesterol was present in the form of a solid wafer (approximately 10mg). Where indicated the reaction contained 10 μmol/ml l-alanine and 10%ethanol. The final volume was 1 ml. After the indicated period ofincubation (22° C.), Aspergillus fumigatus was plated on Sabouraud'sdextrose agar. The results are expressed as the colony forming units(CFU's) counted. ND indicates that the experiment was not done. Theresults are presented in Table 21.

                                      TABLE 21    __________________________________________________________________________    Effects of l-Alanine on Cholesterol Oxidase-Haloperoxidase Microbicidal    Action Against Aspergillus fumigatus Spores Using Solid Cholesterol    as Substrate With and Without Ethanol:                       A. fumigatus Survival,    Treatment:         CFU (Post Exposure Time in Hours)    Haloperoxidase            l-Alanine                 Ethanol                       0.5 Hr                            1 Hrs.                                  2 Hrs.                                       4 Hrs.    __________________________________________________________________________    None    None 10%   960,000                            1,900,000                                  960,000                                       800,000    None    10 μmol                 10%   1,180,000                            1,220,000                                  820,000                                       158,000    MPO, 10 pmol            None 10%   920,000                            1,540,000                                  1,120,000                                       1,020,000    MPO, 10 pmol            10 μmol                 10%   82,000                            0     0    0    EPO, 10 pmol            None 10%   1,120,000                            3,000,000                                  040,000                                       940,000    EPO, 10 pmol            10 μmol                 10%   0    0     0    0    None    None None  ND   2,300,000                                  ND   980,000    None    10 μmol                 None  ND   1,980,000                                  ND   1,900,000    MPO, 10 pmol            None None  ND   2,380,000                                  ND   1,860,000    MPO, 10 pmol            10 μmol                 None  ND   1,640,000                                  ND   10,000    EPO, 10 pmol            None None  ND   2,620,000                                  ND   1,340,000    EPO, 10 pmol            10 μmol                 None  ND   1,340,000                                  ND   0    __________________________________________________________________________

As shown in Table 21, in the presence of ethanol plus l-alanine, thecholesterol oxidase-MPO formulation effected a tenfold killing of thespores at thirty minutes and complete killing at one, two, and fourhours. L-alanine was required for effective MPO-dependent killing. Inthe presence of ethanol and l-alanine but absence of haloperoxidase,cholesterol oxidase was mildly effective but only after four hoursincubation. The most effective formulation was cholesterol oxidase-EPOwith ethanol and l-alanine. Complete spore killing was observed for alltest intervals.

Inclusion of ethanol greatly increased the effectiveness of all theformulations tested. Sporadical action is faster and more intense in thepresence of 10% ethanol. This ethanol effect may result from increasedsolubilization of the solid cholesterol resulting in increasedcholesterol oxidase activity. The results also demonstrate that 10%ethanol has no detrimental effect on the cholesteroloxidase-haloperoxidase function. Inclusion of an adequate quantity ofethanol or other nontoxic solvent in the formulation also assists inmaintaining sterility of the system in the absence of cholesterol andimproves the shelf-life of the reaction mixture.

Various modifications and adaptations of the methods and compositions ofthe invention will be apparent from the foregoing to those skilled inthe art. Any such modifications and adaptations are intended to bewithin the scope of the appended claims except insofar as precluded bythe prior art.

I claim:
 1. A method for killing or inhibiting the growth of sporulatingmicroorganisms selected from the group consisting of bacteria, yeast orfungi, in a subject in need thereof, comprising:administering, in thepresence of a halide selected from the group consisting of chloride orbromide, an antimicrobially effective amount of a composition comprisinga haloperoxidase and at least one agent of the formula: ##STR5## whereinR₁ is hydrogen, an unsubstituted, or hydroxy or amino substituted,straight or branched chain alkyl group having from 1 to 6 carbon atoms,and R₂ is hydrogen or a straight or branched chain alkyl group havingfrom 1 to 6 carbon atoms; wherein said composition is administered bydirectly contacting said composition with the sporulating microorganism.2. The method of claim 1 wherein the haloperoxidase is selected from thegroup consisting of myeloperoxidase, eosinophil peroxidase andcombinations thereof.
 3. The method of claim 2 wherein thehaloperoxidase is myeloperoxidase.
 4. The method of claim 3 wherein thecomposition comprises at least 0.01 pmol/ml of myeloperoxidase in aliquid carrier.
 5. The method of claim 4 wherein the compositioncomprises from 0.1 pmol/ml to 500 pmol/ml of myeloperoxidase.
 6. Themethod of claim 4 wherein the composition comprises from 0.5 pmol/ml to50 pmol/ml of myeloperoxidase.
 7. The method of claim 2 wherein thehaloperoxidase is eosinophil peroxidase.
 8. The method of claim 7wherein the composition comprises at least 0.01 pmol of eosinophilperoxidase in a liquid carrier.
 9. The method of claim 8 wherein thecomposition comprises from 0.1 pmol to 500 pmol per ml of eosinophilperoxidase in a liquid carrier.
 10. The method of claim 8 wherein thecomposition comprises from 0.5 pmol/ml to 50 pmol/ml of eosinophilperoxidase.
 11. The method of claim 8 wherein the composition furthercomprises from 10 nmol/ml to 10 μmol/ml of bromide.
 12. The method ofclaim 1 wherein said agent is an α-amino acid selected from the groupconsisting of glycine, alanine, valine, leucine, isoleucine, serine,threonine, lysine, and the alkyl esters of any of the members of thegroup.
 13. The method of claim 12 wherein the composition comprises atleast 0.005 μmol/ml of said agent in a liquid carrier.
 14. The method ofclaim 13 wherein the composition comprises from 0.05 μmol/ml to 50μmol/ml of said agent in a liquid carrier.
 15. The method of claim 1wherein the composition further comprises hydrogen peroxide or anoxidase that produces a peroxide in the presence of a substrate for theoxidase.
 16. The method of claim 15 wherein the composition comprises aperoxide producing oxidase effective to generate from 100 pmol to 50μmol peroxide per ml per minute when in the presence of a substrate forthe oxidase.